The goal of this project is to develop a validated, quantitative assay for the fragile X mental retardation protein (FMRP) to aid in the clinical management of those with fragile X syndrome (FXS), autism and other disorders and to advance FXS research. FXS is the leading form of inheritable intellectual disability, ranging from mild to severe and is the most common known mutation in autism. It is caused by CGG repeat expansion in the X-linked FMR1 gene. In non-affected individuals, 6 to 44 repeats occur in the FMR1 5' UTR, whereas in those with FXS, >200 repeats are observed, causing substantially reduced levels of FMRP and intellectual disability. Expansion to 55-200 repeats results in normal or much smaller reductions in FMRP. These individuals are unaffected or present a varying, less serious degree of cognitive, behavioral and emotional dysfunctions. Individuals with fewer than 55 repeats almost never manifest FMR1-related clinical features. FMRP diagnostic testing is already recognized in the 2013 ACMG fragile X guidelines. However, reliable correlation between FMRP levels and patient diagnosis and prognosis has been undermined by both biological and technical issues. There is a critical need for a well validated and standardized quantitative FMRP assay to support researchers and clinicians in their efforts to understand FXS biology and to aid in patient management. Biological challenges to the reliable interpretation of FMR1 molecular tests include somatic mosaicism, or the presence of varying genotypes and/or methylation states in different tissues. Importantly, case studies have revealed tissue-specific differences for fragile X mutations, wherein buccal cells that share an ectodermal developmental lineage with brain tissue may be more clinically informative than blood. We will test the feasibility of using BCs to assay FMRP because they may better represent fragile X biology in the brain. Technical challenges include pre-analytical limitations, substandard antibodies, and the lack of quantitative FMRP standards. Recently an FMRP assay was published that addresses these issues, including compatibility with dried blood spots (DBS) and a workflow on the Luminex platform that is amendable to routine testing. Our overall objective is to make improvements to this assay by reducing sources of variation, validating the test with annotated clinical samples, and then launching the assay for the analysis of blood and buccal cells stabilized on 903 paper. This technology will enable the accurate quantification of FMRP and FMR1 genotyping from a single sample, thereby simplifying the collection of a comprehensive FXS dataset, enabling diagnostic and prognostic applications, and improving our overall understanding of FXS biology. Specific Aim 1: Complete a multi-site validation of the published assay using common reagents and samples. Specific Aim 2: Develop the protocols and reagents to normalize for the FMRP-producing blood cells in a DBS sample. Specific Aim 3: Demonstrate the feasibility of using buccal cells stored on 903 papers for assaying FMRP, and perform a pilot clinical study.